Serum monovette

Blood samples (taken immediately before and 6, 12, 24, 48, 72, 96, and 120 h after dosage; Serum Monovette^®, Sarstedt AG & Co., Sarstedt, Germany; Figure 5) were kept upright at room temperature for at least 30 min for clotting, subsequently centrifuged at 2123× g for 15 min, and serum aliquots were stored at −20 °C until analysis. After thawing, following blood biochemical parameters were analyzed using a photometer (Eurolyser CCA 180, Eurolyser Diagnostica GmbH, Salzburg, Austria): total blood cholesterol (CHOL, mg/dL), protein (PROT, g/L), albumine (ALB, g/L), alkaline phosphatase (AP, U/L), total bilirubin (BILI, mg/dL), gamma-glutamyl-transferase (γGT, U/L), aspartate-amino transferase (AST, U/L), urea (UREA, mg/dL), and triacylglycerides (TRI, mg/dL). Reagents used for photometric analysis were obtained from Greiner Diagnostic GmbH (Greiner Diagnostic GmbH, Bahlingen, Germany).

Blood samples were taken from the external jugular vein of the sows and from the cranial vein of the piglets [29 (link)]. The blood was collected in a tube containing coagulation factor (Sarstedt Serum Monovette^®, Sarstedt AG & Co. KG, Nümbrecht, Germany). The blood was centrifuged at 3000 rpm for 6 min and the obtained serum was sent to a veterinary diagnostic laboratory (SAN Group Biotech Germany GmbH).

To obtain serum miR-143 levels at the time point of admission to the ICU (before any therapeutic intervention), blood was collected using serum monovettes (Sarstedt, Germany), centrifuged for 8 minutes at 2000 g using a Rotixa 50 centrifuge (Hettich, Germany) following standard protocols within the Labordiagnostisches Zentrum (LDZ) of the university clinic (RWTH) Aachen for patient routine care. No further clearance was performed before RNA isolation. After centrifugation, samples were immediately placed on ice and frozen at -80°C until RNA isolation. Interleukin-6 (IL-6), Interleukin-10 (IL-10), TNF, soluble urokinase plasminogen activator receptor (suPAR), Osteopontin, Glucocorticoid-induced TNF receptor ligand (GITRL), and A proliferation-inducing ligand (APRIL) were measured as described previously (e.g., [16 (link)–20 (link)]). All other laboratory markers mentioned within this manuscript were measured as part of clinical routine at the Labordiagnostisches Zentrum (LDZ) of the University Hospital (RWTH) Aachen. Glomerular filtration rates (GFR) were calculated on basis of serum cystatin C levels. ICU mortality was defined as death on ICU; overall mortality included death at the ICU or during the observation period (after discharge from the ICU and hospital).

Venous blood from four healthy donors was drawn into 7.5 ml serum Monovettes (Sarstedt) and cell-free supernatants were obtained after sedimentation. Serum from all four donors was pooled, and stored at − 80 °C. Written informed consent was obtained according to the local Ethics Committee (reference number A2017-0191) and the guidelines for the use of human material. To assess serum stability of SpyADI and SpyADI-PEG20, 200 µl enzyme solution with 2 mg/ml were mixed with 600 µl human serum and incubated at 37 °C for up to 48 h. As controls, (1.) 200 µl of SpyADI or SpyADI-PEG20 were mixed with 600 µl PBS and (2.) 600 µl serum were mixed with 200 µl PBS and treated equally. At different time points, 30-µl samples were taken, immediately mixed with SDS sample buffer, and stored on ice. Samples were subjected to SDS-PAGE and Western blot analysis with alkaline phosphatase conjugated StrepTactin to detect SpyADI and carboxyterminal degradation fragments. Furthermore, samples were taken for ADI activity measurements.

Blood sampling from adult sows was performed without sedation under manual fixation. Whole blood was drawn from the jugular vein with single‐use needles (Ehrhardt Medizinprodukte, Geislingen, Germany) into lithium heparin and serum Monovettes (Sarstedt, Nümbrecht, Germany). Blood from the baboons was taken using a central venous catheter.

EDTA-anticoagulated whole blood was collected in EDTA monovettes (Sarstedt, Nümbrecht, Germany). Serum samples were collected in serum monovettes (Sarstedt), followed by clotting reaction and centrifugation (4000×g, 10 min). Within two days of collection, samples were stored at −70°C until assayed as described below. Genomic DNA of IE patients was isolated from surgically removed heart valves. Total DNA was extracted with the QIAamp DNA blood kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions, and DNA was eluted with 50 µL sterile distilled water from the QIAamp column.